Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

Abstract

Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation

“…In bacteria, the most important ribosome rescue process is trans -translation, which not only releases the ribosome but also targets the defective mRNA and the incomplete nascent peptide for degradation ( 42 ). In some bacteria, including Escherichia coli and B. subtilis , alternative factors have been found that can release ribosomes but do not eliminate the mRNA or nascent peptide ( 43 – 45 ). For E. coli , a conservative estimate is that 2 to 4% of translation reactions require trans -translation to release ribosomes ( 46 ).…”

Functionally uncoupled transcription–translation in Bacillus subtilis

“…This fundamental difference may contribute to the divergent regulatory mechanisms observed between these species in two major ways. First, it enables intrinsic terminators to generate functional nonstop mRNAs for highly translated genes in Bacilli 32 , such as the mRNA for an alternative ribosome rescue factor whose analogue in E. coli is generated post-transcriptionally by RNase III cleavage 33 , 34 . Second, the lack of coupling likely leads to avoidance of ribosome-controlled transcriptional attenuators and instead favors riboswitch- or protein-based mRNA leaders that are widely observed in B. subtilis 14 , 17 - 19 .…”

Ribosome Rescue Pathways in Bacteria

“…Bacteria have evolved a diverse array of mechanisms to rescue no-go and non-stop ribosomal complexes ( Figure 1 ). This is critical for survival in bacteria since otherwise ribosomes and tRNAs become sequestered from the pool of free translational components and the capacity of the cell to produce proteins rapidly diminishes ( Keiler et al, 1996 ; Karzai et al, 1999 ; Tenson et al, 1999 ; Moore and Sauer, 2005 ; Chadani et al, 2010 , 2011b ; Goralski et al, 2018 ; Shimokawa-Chiba et al, 2019 ). The best characterized bacterial rescue systems that resolve no-go and non-stop ribosomes include trans -translation ( Figure 1C , upper panel), alternative ribosome rescue factors (Arfs) ( Figure 1C , lower panel), the recently identified bacterial ribosome quality control (RQC; Figures 1D,E ) and peptidyl-tRNA drop-off ( Figures 1F,G ; Menninger, 1976 ; Keiler et al, 1996 ; Karzai et al, 1999 ; Chadani et al, 2010 , 2011b ; Handa et al, 2011 ; Goralski et al, 2018 ; Shimokawa-Chiba et al, 2019 ).…”

“…Interestingly, ArfT cooperates with both RF1 and RF2 for hydrolysis of the nascent chain ( Goralski et al, 2018 ). BrfA is likely limited to the Bacillus genus and exclusively recruits RF2, and hence BrfA has some similarities but also differences to ArfA ( Shimokawa-Chiba et al, 2019 ). It is unclear whether an unidentified Arf system exists in S. aureus , however, S. aureus also appears to have the RQC pathway (discussed below; Lytvynenko et al, 2019 ).…”